Match criteria for human cell line authentication: Where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross‐contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN‐0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines—duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50–79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.     

The Safe Urban Harvests Study: A Community-Driven Cross-Sectional Assessment of Metals in Soil,Irrigation Water,and Produce from Urban Farms and Gardens in Baltimore,Maryland

Background: Emerging evidence suggests social, health, environmental, and economic benefits of urban agriculture (UA). However, limited work has characterized the risks from metal contaminant exposures faced by urban growers and consumers of urban-grown produce.Objectives: We aimed to answer community-driven questions about the safety of UA and the consumption of urban-grown produce by measuring concentrations of nine metals in the soil, irrigation water, and urban-grown produce across urban farms and gardens in Baltimore, Maryland.Methods: We measured concentrations of 6 nonessential [arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), lead (Pb), nickel (Ni)] and three essential [copper (Cu), manganese (Mn), zinc (Zn)] metals in soil, irrigation water, and 13 types of urban-grown produce collected from 104 UA sites. We compared measured concentrations to existing public health guidelines and analyzed relationships between urban soil and produce concentrations. In the absence of guidelines for metals in produce, we compared metals concentrations in urban-grown produce with those in produce purchased from farmers markets and grocery stores (both conventionally grown and U.S. Department of Agriculture–certified organic).Results: Mean concentrations of all measured metals in irrigation water were below public health guidelines. Mean concentrations of nonessential metals in growing area soils were below public health guidelines for Ba, Cd, Pb, and Ni and at or below background for As and Cr. Though we observed a few statistically significant differences in concentrations between urban and nonurban produce items for some combinations, no consistent or discernable patterns emerged.Discussion: Screening soils for heavy metals is a critical best practice for urban growers. Given limitations in existing public health guidelines for metals in soil, irrigation water, and produce, additional exposure assessment is necessary to quantify potential human health risks associated with exposure to nonessential metals when engaging in UA and consuming urban-grown produce. Conversely, the potential health benefits of consuming essential metals in urban-grown produce also merit further research. https://doi.org/10.1289/EHP9431     

Lymphedema: experience of a cohort of women with breast cancer followed for 4 years after diagnosis in Victoria, Australia

Purpose

The aim of this work was to study the incidence and prevalence of self-reported lymphedema in breast cancer survivors between 2 and 4 years following diagnosis, the factors associated with the development of lymphedema and the impact of lymphedema on psychological well-being.

Methods

We assessed self-reported lymphedema in the BUPA Health Foundation Health and Wellbeing After Breast Cancer Study, a questionnaire-based study of 1,683 women newly diagnosed with their first episode of invasive breast cancer in Victoria, Australia. Psychological well-being was assessed using the Psychological General Well-being Index.

Results

Two years after diagnosis, nearly 20 % of women reported lymphedema and this proportion remained above 18 % 2 years later. However, self-reported lymphedema was a dynamic phenomenon, with the condition resolving in some women and others reporting onset for the first time up to 4 years from diagnosis. Lymphedema 2 years from diagnosis was positively associated with the number of nodes removed at initial surgery, although this variable only explained a small proportion of the likelihood of reporting lymphedema. The presence of lymphedema was associated with lower psychological general well-being.

Conclusions

Lymphedema after breast cancer treatment frequently has a dynamic pattern and may emerge as an issue for women several years after their initial treatment. It is associated with a lower level of general well-being.     

<Superscript>18</Superscript>F–FDG-PET/CT for systemic staging of patients with newly diagnosed ER-positive and HER2-positive breast cancer

Objectives

This study assesses ¹⁸F–FDG-PET/CT for patients with newly diagnosed estrogen receptor-positive/human epidermal growth factor receptor-negative (ER+/HER2-) and human epidermal growth factor receptor-positive (HER2+) breast cancer.

Methods

In this Institutional Review Board-approved retrospective study, our Healthcare Information System was screened for patients with ER+/HER2- and HER2+ breast cancer who underwent ¹⁸F–FDG-PET/CT prior to systemic or radiation therapy. The initial stage was determined from mammography, ultrasound, magnetic resonance imaging, and/or surgery.¹⁸F–FDG-PET/CT was evaluated to identify unsuspected extra-axillary regional nodal and distant metastases. The proportion of patients upstaged overall and stratified by stage and receptor phenotypes was calculated along with confidence intervals (CI).

Results

A total of 238 patients with ER+/HER2- and 245 patients with HER2+ who met inclusion criteria were evaluated. For patients with ER+/HER2-breast cancer, ¹⁸F–FDG-PET/CT revealed unsuspected distant metastases in 3/71 (4%) initial stage IIA, 13/95 (14%) stage IIB, and 15/57 (26%) stage III. For patients with HER2+ breast cancer, ¹⁸F–FDG-PET/CT revealed unsuspected distant metastases in 3/72 (4%) initial stage IIA, 13/93 (14%) stage IIB, and 13/59 (22%) stage III. The overall upstaging rate for IIB was 14% (95% confidence interval (CI): 9–20%).

Conclusions

¹⁸F–FDG-PET/CT revealed distant metastases in 14% (95% CI: 9–20%) of patients with stage IIB ER+/HER2- and HER2+ breast cancer, which is similar to upstaging rates previously seen in patients with stage IIB triple-negative breast cancer (15%, 95% CI: 9–24%). The detection of unsuspected distant metastases in these patients alters treatment and prognosis. NCCN guidelines should consider adding patients with stage IIB breast cancer for consideration of systemic staging with ¹⁸F–FDG-PET/CT at the time of initial diagnosis.

     

Content Analysis and Key Informant Interviews to Examine Community Response to the Purchase, Possession, and/or Use of Tobacco by Minors

The aim of this study was to identify and describe local ordinances in New Jersey that make it illegal for minors to purchase, possess, and/or use tobacco (PPU). A coding instrument was formulated and content analysis of each ordinance was conducted between March 1999 andJanuary 2002. Additionally, key informant interviews with community officials were conducted by telephone between September 2000 and February 2002 to collect qualitative information on implementation and enforcement. Content analysis of identified ordinances assessed when the ordinance was enacted, specific laws and clauses included, enforcing party, area of jurisdiction, and penalties associated with a citation. Key informant interviews assessed the catalyst for enacting the ordinance, penalties, enforcement activity, and method of tracking citations. As of January 2002, 48 municipalities in New Jersey had passed mandates banning minor purchase, possession, and/or use of tobacco. Of the 48 ordinances reviewed, 71% were passed during or after 1998. Nearly all of the ordinances (94%) included prohibited minor usage of tobacco, 77% prohibited minor possession of tobacco and 23% prohibited minor purchase of tobacco. In over 80% of communities, municipal police departments were responsible for enforcement. Two out of 35 communities reached for interview reported having a formal system for tracking enforcement or citations. The results illustrate that local PPU ordinances in New Jersey vary widely both in principle and in practice, suggesting that such ordinances may be too heterogeneous and lacking in cohesion to have any impact on youth smoking.     

Antagonism of innate immunity by paramyxovirus accessory proteins

Paramyxovirinae, a subfamily of Paramyxoviridae, are negative strand RNA viruses comprised of many important human and animal pathogens, which share a high degree of genetic and structural homology. The accessory proteins expressed from the P/V/C gene are major factors in the pathogenicity of the viruses, because of their ability to abrogate various facets of type I interferon (IFN) induction and signaling. Most of the paramyxoviruses exhibit a commonality in their ability to antagonize innate immunity by blocking IFN induction and the Jak/STAT pathway. However, the manner in which the accessory proteins inhibit the pathway differs among viruses. Similarly, there are variations in the capability of the viruses to counteract intracellular detectors (RNA helicases, mda-5 and RIG-I). Furthermore, a functional specificity in the antagonism of the IFN response has been reported, suggesting that specificity in the circumvention of innate immunity restricts viral host range. Available evidence indicates that paramyxoviruses employ specific strategies to antagonize the IFN response of their specific hosts, which is one of the major factors that determine viral pathogenicity and host range.     

<Superscript>18</Superscript>F-FDG-PET/CT for systemic staging of newly diagnosed triple-negative breast cancer

Purpose

National Comprehensive Cancer Network guidelines recommend ¹⁸F-FDG-PET/CT, in addition to standard staging procedures, for systemic staging of newly diagnosed stage III breast cancer patients. However, factors in addition to stage may influence PET/CT utility. As breast cancers that are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor (triple-negative breast cancer, or TNBC) are more aggressive and metastasize earlier than other breast cancers, we hypothesized that receptor expression may be one such factor. This study assesses ¹⁸F-FDG-PET/CT for systemic staging of newly diagnosed TNBC.

Methods

In this Institutional Review Board-approved retrospective study, our Healthcare Information System was screened for patients with TNBC who underwent ¹⁸F-FDG-PET/CT in 2007–2013 prior to systemic or radiation therapy. Initial stage was determined from mammography, ultrasound, magnetic resonance imaging, and/or surgery, if performed prior to ¹⁸F-FDG-PET/CT. ¹⁸F-FDG-PET/CT was evaluated to identify unsuspected extra-axillary regional nodal and distant metastases, as well as unsuspected synchronous malignancies. Kaplan Meier survival estimates were calculated for initial stage IIB patients stratified by whether or not stage 4 disease was detected by ¹⁸F-FDG-PET/CT.

Results

A total of 232 patients with TNBC met inclusion criteria. ¹⁸F-FDG-PET/CT revealed unsuspected distant metastases in 30 (13 %): 0/23 initial stage I, 4/82 (5 %) stage IIA, 13/87 (15 %) stage IIB, 4/23 (17 %) stage IIIA, 8/14 (57 %) stage IIIB, and 1/3 (33 %) stage IIIC. Twenty-six of 30 patients upstaged to IV by ¹⁸F-FDG-PET/CT were confirmed by pathology, with the remaining four patients confirmed by follow-up imaging. In addition, seven unsuspected synchronous malignancies were identified in six patients. Initial stage 2B patients who were upstaged to 4 by ¹⁸F-FDG-PET/CT had significantly shorter survival compared to initial stage 2B patients who were not (3-year Kaplan Meier estimate 0.33, 95 % CI 0.13-0.55 versus 0.97, CI 0.76-0.93, p?<?.0001).

Conclusion

F-FDG-PET/CT revealed distant metastases in 15 % of patients with stage IIB TNBC. Stage IIB patients upstaged to 4 by ¹⁸F-FDG-PET/CT had significantly shorter survival than those who were not, consistent with ¹⁸F-FDG-PET/CT detecting an increased burden of disease. This study provides further evidence that populations of patients with stage IIB breast cancer, such as TNBC, should be considered for systemic staging with ¹⁸F-FDG-PET/CT at the time of initial diagnosis.

     

Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry

Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from volunteer buccal swabs but the system has not been tested using more challenging sample types. For mass spectrometry to be considered as a valid alternative to Sanger sequencing it must also be demonstrated to be suitable for use with more limiting sample types such as old teeth, bone fragments, and hair shafts. In 2009 the Commonwealth War Graves Commission launched a project to identify the remains of 250 World War I soldiers discovered in a mass grave in Fromelles, France. This study characterises the performance of both Sanger sequencing and the T5000 platform for the analysis of the mitochondrial DNA extracted from 225 of these remains, both in terms of the ability to amplify and characterise DNA regions of interest and the relative information content and ease-of-use associated with each method.     

Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes

BackgroundCoumarin anticoagulants such as warfarin are used to treat and prevent thromboembolic events in patients. The required dosage is difficult to predict and the risk of over or under anticoagulation are dependent on several environmental and clinical factors, such as concurrent medication, diet, age and genotype for polymorphisms in two genes CYP2C9 and VKORC1.MethodsA novel fluorescent PCR genotyping assay using HyBeacon® probes, was developed to enable clinical staff to genotype the CYP2C9*2 and CYP2C9*3 alleles and the VKORC1 G-1639A polymorphism directly from unextracted blood samples. A prototype PCR instrument, Genie 1, suitable for point of care use was developed to carry out the assays. The panel of tests was validated by analysing blood samples from 156 individuals and comparing genotypes with data obtained using DNA samples from the same individuals. The accuracy of genotypes obtained with the Genie 1 was compared against results from well validated real time PCR and PCR-restriction fragment length polymorphism analysis.ResultsIdentical results were obtained for the newly developed HyBeacon® method and the validation method in all cases except for one where no result was obtained for the VKORC1 polymorphism on the Genie instrument. The samples used for validation represented all six possible *2 and *3 allele-related CYP2C9 genotypes and all three VKORC1 G-1639A genotypes.ConclusionsWe observed excellent accuracy for the newly developed method which can determine genotype in less than 2 h.     

Human parainfluenza virus type 1 but not Sendai virus replicates in human respiratory cells despite IFN treatment

Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN-beta into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-beta, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN-beta pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV.